Pink-flowered, high protein soybean plants and methods for their production

ABSTRACT

A pink-flowered soybean plant which possesses large seeds and high protein seed content, without a concomitant decrease in yield. The pink-flowered soybean plant is easily distinguished from wild-type and domesticated cultivars by virtue of its unique flower color and appearance. The two commercially significant traits, large seed size and high protein seed content, cosegregate with the pink flower trait. Seed size and protein content of soybean cultivars are therefore efficiently and quickly increased by crossing with the pink-flowered plant, then selecting for pink-flowered progeny. Prior difficulties in tracking and monitoring increases in seed protein content are eliminated.

BACKGROUND OF THE INVENTION

The present invention relates to a novel pink-flowered soybean plantpossessing the associated phenotypes of high protein seed content, highyield, and large seeds. A novel soybean plant is disclosed, easilydistinguished from wild-type and domesticated cultivars in having a pinkflower color. More particularly, this novel pink-flower phenotype isassociated with an increased seed size and an increased seed proteincontent without a concomitant decrease in yield, two attributes ofcommercial significance. A method of plant breeding, for introducing thepink flower color, increased seed size and increased seed protein traitsinto domestic cultivars is also disclosed. Further, a process to produceand select for large seeded, high protein soybean varieties, based onthis novel flower color, is disclosed.

Soybean (Glycine max (L.)) has become an important part of theagricultural economy due to its use as a source of protein. High proteinsoybean varieties are especially in demand both in the domestic andexport markets.

A goal in nearly every soybean improvement program, therefore, is toincrease the protein content in the seed. Protein is affected by manygenes and can be increased through traditional breeding practices,however improvement will be slow. A minimum of seven years andconsiderable resources are needed to develop improved varieties. Varietydevelopment through normal plant breeding techniques requires constantmonitoring of seed protein content, typically with near-infraredreflectance. In addition, protein increase is usually associated with ayield decrease, making it difficult to maintain or increase yield whileimproving protein content.

Of particular importance to the soybean breeder is the demand for alarge seeded, high protein variety. Large seeded soybean varieties areespecially desirable for sale to the specialty soybean export market.Large bean size is important, for example, for soybeans used in themanufacture of tofu.

A need therefore exists for a high-yield soybean variety having largeseeds and high protein content, and methods for introducing thesedesired traits into domestic cultivars. A need also exists for aselection method whereby hybrid progeny possessing these desiredagronomic traits are readily identified, eliminating the need forconstant monitoring of seed protein content.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a novelsoybean variety exhibiting the combined phenotypes of large seed size,high protein seed content and high yield. Unexpectedly, the soybeanplants of the present invention generate elevated levels of seedprotein, without the generally associated decrease in yield.

It is another object of the present invention to provide a large seeded,high protein, high yield soybean variety which is easily distinguished,on the basis of flower color, from other soybean varieties. The presentinvention is based in part on the recognition that these desiredagronomic traits, large seed size and increased protein content,co-segregated with a novel flower color.

It is a further object of the present invention to provide a largeseeded, high protein, high yield soybean plant which exhibits the novel,pink flower phenotype.

It is yet another object of the present invention to provide new soybeanplants that can be used efficiently to produce parent lines and hybridspossessing desirable agronomic traits.

The present invention also comprises a process for breeding the novelpink flower color, enhanced seed size, and increased seed protein traitsinto wild-type soybeans and domestic cultivars.

Finally, the present invention comprises a practical and efficientmethod for producing and selecting for large seeded, high protein, highyield soybean varieties, based on the novel pink flower color. A soybeanbreeder can increase seed size and protein content efficiently andquickly by crossing with the pink flowered soybean variety of thepresent invention, then selecting for plants with pink flowers.

In accomplishing the foregoing objects, there has been provided, inaccordance with the present invention, a pink-flowered soybean linewhich has an increased seed protein content of approximately 1.5percentage points, relative to the normal seed protein content ofcommercial cultivars.

There has also been provided in accordance with the present invention apink-flowered soybean line which has an increased seed size ofapproximately 25%, relative to the normal seed size of commercialcultivars.

The breeding and selection methods described herein are applicable toall soybean varieties, including wild-type and commercial cultivars. Thepink flower phenotype is controlled by a single recessive gene, whichsegregates according to normal Mendelian genetic principles. Therefore,conventional plant breeding techniques may be used to introduce thephenotypes of enlarged seed size and increased seed protein into anysoybean variety using the novel flower color as a marker. The presentinvention is exemplified by its application to soybean (Glycine max);however, its operating principles may be applied to other species ofsoybean. The invention is not limited to any particular soybeancultivar, but may be applied generally to any plant variety of the genusGlycine, whether wild, domestic or hybrids of the two. The term soybeanis used herein to denote the species Glycine max and all domesticcultivars thereof.

Other objects, features, and advantages of the present invention willbecomes apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of the present description, the terms "cultivar" and"variety" are used synonymously to refer to a group of plants within aspecies (e.g., Glycine max) which share certain constant characters thatseparate them from the typical form and from other possible varietieswithin that species. While possessing at least one distinctive trait, avariety is also characterized by a substantial amount of overallvariation between individuals within the variety, based primarily on theMendelian segregation of traits among the progeny of succeedinggenerations. A "line," as distinguished from a "variety," denotes agroup of plants which display less variation between individuals,generally (although not exclusively) by virtue of several generations ofself-pollination. In addition, a "line" is defined, for the purpose ofthe present invention, sufficiently broadly to include a group of plantswhich carry a mutation for a particular trait, specifically the pinkflowered trait as disclosed herein.

As used herein, the terms "co-segregate," "hybrid," "crossing,""recovering progeny," and "selfing," refer to their conventionalmeanings as understood in the art (see, for instance, Knowles, P. F. andF. N. Briggs, Introduction to Plant Breeding (Reinhold PublicationCorp., New York, N.Y., 1967). "Substantial" is defined as statisticallysignificant.

Most soybean cultivars have either purple or white flowers. Woodworth,C. M. (1923) "Inheritance of Growth Habit, Pod Color, and Flower Colorin Soybeans," J Am. Soc'y Agron 15:481-495. Hartwig, E. E. and K. Hinson(1962) "Inheritance of Flower Color in Soybeans," Crop Sci. 2:152-153,described dilute-purple and near-white flowers, but these flowers appearwhite and identification requires close examination of the separatedstandard. In addition, Buzzel, R. I., et al. (1977) "Inheritance andLinkage of a Magenta Flower Gene in Soybeans," Can. J. Genet. Cytol.19:749-751, report a magenta flower color mutant.

A description of a new flower color, denoted as pink, is presented byStephens, P. A. and C. D. Nickell (1991) "A Pink Flower Color Mutant inSoybean," Soybean Genet. Newsl 18:226-228, This pink flower was firstobserved during the summer of 1989 in two F4:5-derived plant rows(designated LN89-5320 and LN89-5322) segregating for purple and pinkflower pigmentation while homozygous for gray pubescence and imperfectblack hilum. These lines had originated from the cross (`Sherman` x`Asgrow A2943`) x `Elgin 87`. All F4 plant selections from this crosswere purple in flower color. For the two F4:5 plant rows having the pinkflower mutation, F5 plants in both rows were classified as pink orpurple in flower color, harvested individually, and planted in thespring of 1990. To determine if the pink flower trait is different inappearance from known color genes, pink flowered plants were plantedalongside `Clark` and `Harosoy` flower color isolines (Bernard, 1978;Table 2).

The pink flower color disclosed herein is quite distinct from allpreviously reported flower colors. Pink flowers are uniform in colorthroughout the petals, in contrast to purple, magenta, dilute-purple,and near-white flowers which have increased pigmentation in the veinswhen compared to the interveinal areas. "Pink" flowers, as used herein,refers to soybean flowers which appear pink in color and uniform inpigmentation throughout the petals, as distinguished from the soybeanflowers of known varieties.

The novel soybean plant of the present invention exhibits, among otherthings, a unique flower color. The gene controlling the pink flowertrait is not allelic to any of the known flower color genes. Flowercolor segregation data for F₂ plants demonstrates that the pink flowertrait is novel, is not linked to other flower color genes, and is notinfluenced by cytoplasm (Table 5). Segregation data for selfed progenyindicates that the pink flower trait is controlled by a single recessivegene (Table 3). Selfed progeny from ten heterozygous rows, classified assegregating 3:1 (purple:pink) for flower color (Stephens and Nickell,.Supra), segregated 5:3 (purple:pink) as expected for single recessivegene inheritance. When this pink-flowered plant was crossed with allreported flower color genes and F₂ populations classified for flowercolor (Table 5), results indicated that the pink flower gene wasindependent of known flower color genes and acted as a modifier gene todecrease pigment expression at the W1 locus, required for purple flowerpigmentation, thereby reducing normal purple pigmentation to pink(Stephens and Nickell, supra). The pink flower gene thus interacts withother flower color genes to dilute the intensity of their expression. Asused herein, the gene symbol wp means pink flower color inheritance.

As noted above, the pink flowered soybean plant of the present inventionwas crossed with all known flower color genes and the F₂ generationclassified according to flower color. The results are shown in Table 5.When W1-wpwp is in combination with wmwm (magenta) the phenotype appearspink, in combination with W3-w4w4 (purple-throat) the phenotype appearsnear-white, and in combination with w3w3w4w4 (near-white) the flowersappear white. This interaction is demonstrated in the three genesegregation model for the cross LN89-5322-2 X L70-4422. Contributing tothe theoretical 6/64 near-white class are 3/64 W3-w4w4wpwp individualswhich are phenotypically difficult to distinguish from the 3/64w3w3w4w4Wp individuals, and the 1/64 white class appears due to thew3w3w4w4wpwp combination.

The novel soybean line of the present invention is further distinguishedfrom other soybean varieties on the basis of hypocotyl pigmentation.Hypocotyl and flower color pigmentation are closely associated (Hartwigand Hinson, supra). Soybean plants having purple and magenta flowershave purple hypocotyls, and purple-throat types have dilute-purplehypocotyls. The other flower color types, including pink, lack purplepigmentation in the hypocotyl when grown under a 14 hour photoperiod.Anthocyanin malvidin is the predominant pigment responsible for purplecoloration in the hypocotyls and is the final product in the anthocyaninpathway (Peters, D. W., et al. (1984) "Hypocotyl Pigments in Soybeans,"Crop Sci. 24:237-239). The pink phenotype therefore likely represents achange in the anthocyanin production pathway resulting in a lower levelof pigment intensity.

In addition to the unique pink flower color, the novel soybean plant ofthe present invention possesses high protein seed content, high yield,and large seeds. Seed size and protein content have not been previouslyfound to be associated, and association of flower color with agronomictraits has never been reported. It is interesting and most surprising,therefore, to note that the two agronomic traits identified herein,large seed size and high protein seed content, co-segregate with thenovel pink flower trait. The association of these two traits with pinkflower color creates a unique situation where the plant breeder canselect for improved soybean varieties based on flower color. A primaryadvantage of the present invention is therefore the ease with whichplant breeders can follow the pink flower trait, along with theassociated increases in seed size, protein content, and yield.

It is further surprising and unexpected that the increased seed proteincontent, associated with the pink flower trait of the present invention,it not accompanied by a decrease in yield. Protein increase is usuallyassociated with a yield decrease, making it difficult to maintain orincrease yield while improving protein content. Thus, the discovery thatthese two phenomena are not synchronous with respect to thepink-flowered soybean represents yet another advantage of the presentinvention.

Soybean varieties possessing specific, desirable traits are oftendeveloped by traditional plant breeding techniques. Two cultivars aretypically selected for particular traits and then interbred, onecultivar being employed as male and the other as female. After the firstcross, the F₂ generation plants are normally screened for the traits ofinterest. Seeds are saved from the F₂ plants selected and subsequentgenerations are grown up, again selecting desirable plants from eachgeneration. In the case of variety development for increased seedprotein, a minimum of seven years and considerable resources are neededto develop improved varieties. Protein content must be constantlymonitored by analyzing the seed, typically with near-infraredreflectance. In addition, yield decrease is usually associated with aprotein increase, making it difficult to maintain or increase yieldwhile improving protein content.

A large seeded, high protein soybean plant possessing the novel pinkflower color has been isolated and identified. Progeny plants(designated LN89-5320 and LN89-5322) possessing the pink flower colorwere selected and their seeds analyzed for both size and proteincontent. The seed produced by the pink-flowered progeny possess a 25%increase in size and a 1.5 percentage point increase in protein contentrelative to their purple-flowered counterparts (Table 1). Proteinpercentage is the percent by weight of the total dry weight of the seed,as measured by near-infrared reflectance. Seed size is measured in cg,based on the average seed weight of a 100 seed sample.

A pink-flowered plant was crossed with each of the five Clark flowercolor isolines (Bernard, R. L. (1978) "Notice of Release of Clark andHarosoy Isolines," Soybean Genet. Newsl. 1:66-75). A pink-flowered, graypubescence plant served as the female while Clark isolines were used asthe male parent. Although the pink-flowered plant was employed as thefemale, the Clark isolines could have been employed as the female andthe pink-flowered plant could have served as the male. The presentinvention encompasses these alternative possibilities.

Progeny plants possessing the pink flower color are selected and theirseeds analyzed for both size and protein content. The seed produced bythe pink-flowered progeny retain the desired traits, i.e., a 25%increase in size and a 1.5 percentage point increase in protein content.The pink-flowered soybean line of the present invention is usedefficiently to produce parent lines and hybrids possessing the desiredagronomic traits.

The breeding and selection methods described herein are applicable toall soybean varieties, including wild-type and commercial cultivars. Thepink flower phenotype is controlled by a single recessive gene, whichsegregates according to normal Mendelian genetic principles.Conventional plant breeding techniques can therefore be used tointroduce the phenotypes of large seed size and high protein seedcontent into any soybean variety. Thus, by conventional plant breedingtechniques, the ordinary artisan can cross a pink-flowered soybean plantwith any variety to produce a soybean line possessing the desiredphenotypes. The present invention is exemplified by its application tosoybean (Glycine max); however, its operating principles may be appliedto other species of soybean. The invention is not limited to anyparticular soybean cultivar, but may be applied generally to any plantvariety of the genus Glycine, whether wild, domestic or hybrids of thetwo. Although the instant invention is applicable to all soybeanvarieties, the breeding and selection methods are preferably carried outby crossing the pink-flowered soybean line with varieties possessingother valuable agronomic traits, including varieties specificallyadapted to the particular climate area. Soybean varieties are widelyavailable in commerce from several manufacturers, providing progenitorstrains suitable for all climate conditions.

The present invention thus provides a practical and efficient method forproducing and selecting for large seeded, high protein, high yieldsoybean plants, based on the characteristic pink flower color. A soybeanbreeder can increase seed size and protein content efficiently andquickly by crossing with the pink flowered soybean line of the presentinvention, then selecting for plants with pink flowers. Thepink-flowered soybean plant produced by the novel breeding process is apart of this invention. The seeds and progeny plants produced bycrossing with the pink-flowered soybean line are also a part of thisinvention.

Specific aspects and features of the present invention will become moreclear from consideration of the following examples which are set forthto further illustrate the principles of the invention and are notintended, in any way, to be limitative thereof.

EXAMPLES EXAMPLE 1 Soybean Breeding and Selection Methods

The desired phenotypes of the novel pink-flower soybean, i.e., largeseed size and high protein seed content, are transferred to otherwild-type or commercial cultivars by conventional plant breeding methodsto achieve a new variety combining these desired phenotypes with othervaluable agronomic traits. A soybean plant possessing the pink flowertrait is crossed, for example, with a variety specifically adapted tothe particular climate area.

The desired wild-type soybean, commercial cultivar, or hybrid thereof iscrossed by conventional plant breeding methods with a soybean planthaving the pink flower phenotype. Breeding methods used in accordancewith the present invention include, for example, methods described inKnowles, P. F. and F. N. Briggs, Introduction to Plant Breeding(Reinhold Publication Corp., New York, N.Y., 1967), incorporated hereinby reference, or any like methods known in the art. Hybrid progenyexhibiting the pink flower trait are selected; seeds from these hybridprogeny will have increased size and protein content. Thus, the seedsize and protein content of any soybean variety is efficiently andquickly increased by crossing with the pink flowered soybean line, thenselecting for pink flowers.

Seeds of Glycine max LN89-5322-1, LN89-5322-2 and LN89-5322-3 weredeposited with the American Type Culture Collection (ATCC), Rockville,Md. Seeds of Glycine max LN89-5322-2 were deposited on Oct. 22, 1993, asATCC 75579. Seeds of Glycine max LN89-5322-1 and LN89-5322-3 weredeposited on Apr. 5, 1995, as ATCC 97108 and ATCC 97109, respectively.

EXAMPLE 2 Agronomic Performance of the Pink-Flowered Soybean

Soybean plants described in Stephens, P. A. and C. D. Nickell (1991) "APink Flower Color Mutant in Soybean," Soybean Genet. Newsl. 18:226-228,were individually harvested, allowed to mature, and additional seedswere collected. Twelve progeny lines were selected for performanceevaluation. Of these twelve lines, several were heterozygous for flowercolor while others were homozygous for either purple or pink flowercolor. The cultivars "Kenwood" (Cianzio, S. R., et al. (1990)"Registration of Kenwood Soybean," Crop Sci. 30:1162), "Burlison"(Nickell, C. D., et al. (1990) "Registration of Burlison Soybean," CropSci 30:232), and "Resnik" (McBlain, B. A., et al (1990) "Registration ofResnik Soybean," Crop Sci. 30:424-425) were included as standards for atotal of 15 entries. Entries were planted in 4-row plots in a randomizedcomplete block design. Each location consisted of two blocks with eachentry entered once per block. Plots were 3 m long with a 76-cm spacingbetween rows. The two center rows were harvested and seed yield wasadjusted for 13% moisture. The eight traits studied were (i) yield (Kgha⁻¹), (ii) plant height at harvest (cm), (iii) maturity (date when atleast 95% of the plants have mature pod color), (iv) lodging (scored onthe basis of 1=all plants erect to 5=all plants prostrate), (v) 100-seedweight (g), (vi) seed quality (on a scale of 1=good to 5=poor), (vii)percentage seed protein, and (viii) percentage seed oil. Protein and oilcomposition data were collected at the USDA Northern Regional ResearchCenter, Peoria, Ill. To obtain oil and protein percentages of the seeds,two replications from each location were composited, then approximately7 g of the composited sample was placed in a Thelco forced air oven for3 hours at 130° C. The seeds were then transferred to 50-g bottles,sealed, and allowed to cool for 1 hour. Samples were then ground in aVarco model MX-228 electric dry-food grinder and returned to the 50-gbottles. The ground meal was analyzed by near-infrared reflectance in aPacific-Scientific feed-grain analyzer. Prior to sample processing, theanalyzer was calibrated with two sealed standards (wheatmeal andsoymeal) and a freshly ground soymeal standard.

The following table presents agronomic data on the pink and purpleflower soybean lines, compared with two soybean cultivars. It can beseen that the seeds from pink flowered plants show a 25% increase insize and a 1.5 unit increase in protein content relative to their purplecounterparts; no statistically substantial decrease in yield is observedwith the increased seed protein. It will be understood that the numberof additional biochemical and physiological traits associated with thepink flower phenotype is not exhausted by the present data disclosedherein. Therefore, any additional traits found to be associated with thepink flower phenotype is deemed to fall within the scope of the claims.

    TABLE 1      Agronomic performance of pink and purple flower soybean lines compared     with two soybean cultivars at four locations in Illinois. 1991. Seed(a)     Seed(b)  Yield Matur- Lodging(a) Height quality weight protein oil Cruse U     rbana G. City Dekalb Entry kg/ha rank ity score cm score cg % % Yield     rank Yield rank Yield rank Yield rank       (c)LN89-5322-1 PkG 3520 8 Sept. 13 1.9 85 1.6 19.9 41.9 20.6 3907 2     3607 6 3720 3 2940 8 (c)LN89-5322-2 PkG 3733 2 Sept. 12 1.6 86 1.7 20.9     41.1 20.9 3607 7 4313 1 3667 5 3460 5 (c)LN89-5322-3 PkG 3560 6 Sept. 13     1.7 84 1.7 20.1 41.6 20.5 3520 8 3767 5 3690 4 3393 6 Burlison 3907 1     Sept. 8 1.1 74 2.0 19.2 42.6 19.5 4073 1 4000 3 3633 6 4047 1 (d)LN89-532     2-6 PG 3733 2 Sept. 10 1.7 89 1.6 16.9 39.9 21.3 3820 4 3913 4 3627 7     3707 2 (d)LN89-5322-11 PG 3533 7 Sept. 11 1.7 87 1.4 16.1 40.6 20.6 3773     6 3380 8 3807 1 3293 1 (d)LN89-5320-5 PG 3680 4 Sept. 10 1.7 83 1.6 15.9     39.5 20.8 3793 5 4120 2 3440 8 3487 4 Kenwood 3660 5 Sept. 9 1.5 84 1.9     14.8 38.5 21.1 3840 3 3593 7 3747 2 3580 3 Average 3666  Sept. 11 1.6 84     1.7 17.9 40.7 20.6 3792  3837  3665  3488 LSD (.05) 327  2 ns 5 0.3 2.0     1.3 0.7 533  720  433  ns CV % 9.2  0.2 30.8 5.7 15.5 5.2 1.5 1.7 6.6     8.8  5.7  11.7 Burlison 3907  Sept. 8 1.1 74 2.0 19.2 42.6 19.5 4073     4000  3633  4047 Kenwood 3660  Sept. 9 1.5 84 1.9 14.8 38.5 21.1 3840     3593  3747  3590 (e)LN89-5322-2 PkG 3733  Sept. 12 1.6 86 1.7 20.9 41.1     20.9 3607  4313  3667  3460 Pink (average) 3604  Sept. 12 1.7 85 1.7     20.3 41.5 20.6 3678  3896  3689  3264 Purple (average) 3649  Sept. 10     1.7 86 1.5 16.3 40.0 20.9 3796  3804  3624  3496     (a)Score: Lodging is rated at maturity: 1 = all plants erect to 5 = all     plants laying flat on soil surface. Seed quality is rated considering the     amount and degree of wrinkling, defective seed coat (growth cracks),     greenishness, and moldy or rotten seeds: 1 = very good to 5 = very poor.     (b)Average of two locations, Urbana and Dekalb, in Illinois. 1991. Seed     weight in cg based on 100 seed sample from each replication. Seed     composition is measured on samples submitted to the USDA Northern Regiona     Research Center, Peoria, IL. A 7 g sample of clean seed is prepared by     compositing an equal volume or weight of seed from each replication withi     a location. Protein and oil percentages (dry weight) are measured using     nearinfrared reflectance.     (c)Pink flower soybean lines.     (d)Purple flower soybean lines.     (e)Pink flowered soybean line being increased during 1992 for possible     release.

EXAMPLE 3 Characterization of the Pink-Flowered Trait in Soybean

Purple and pink-flowered soybean plants described in Stephens, P. A. andC. D. Nickell (1991) "A Pink Flower Color Mutant in Soybean," SoybeanGenet Newsl 18:226-2282, were individually harvested. Seeds of thesepurple and pink-flowered soybean plants were planted alongside "Clark"and "Harosoy" flower color isolines (Bernard, R. L. (1978) "Notice ofRelease of Clark and Harosoy Isolines," Soybean Genet. Newsl. 1:66-75)for comparison of color and appearance. None of the Clark and Harosoyflower color isolines resembled the pink-flowered soybean. The resultsof the comparative color evaluation are set out in the Table 2.

                  TABLE 2                                                         ______________________________________                                        Clark and Harosoy isolines for flower color.                                  Designation  Genotype.sup.  Phenotype                                         ______________________________________                                        Clark 63     W1 w3 W4 Wm    purple                                            L70-4422     W1 W3 w4 Wm    purple throat.sup.                                L68-1774     W1 w3 w4 Wm    near-white                                        L72-2181     W1 w3 W4 wm    magenta                                           L69-4776     w1 w3 W4 Wm    white                                             Harosoy 63   W1 w3 W4 Wm    purple                                            L72-1078     W1 W3 w4 Wm    purple throat                                     L72-1138     W1 w3 w4 Wm    near-white                                        T235         W1 w3 W4 wm    magenta                                           L64-2139     W1 w3 W4 Wm    white                                             ______________________________________                                         .sup. Lines are homozygous for flower color genotype                          .sup. Purple throat = dilutepurple                                       

Table 3 presents segregation data for flower color. The 16 F_(4:5)purple plants segregated in a 1:2 ratio (homozygous:heterozygous) asexpected and the 10 heterozygous rows segregated 3:1 (purple:pink) asexpected for single gene inheritance. Segregation for flower color thusconfirmed that the pink flower color is controlled by a single gene.

                  TABLE 3                                                         ______________________________________                                        Combined flower color data for LN89-5320 and LN89-5322.                                                       Chi                                                    Flower color           square                                                        seg.                  proba-                                           purple no.    pink     Ratio bility                                  ______________________________________                                        1989 plants                                                                              16              7(4 + 3)                                                                             3.1.sup.                                                                            0.55                                             (9 + 7).sup.§           (0.63,                                                                        0.72)                                 1990 F.sub.4:6                                                                           6        10      7     1:2:1.sup.                                                                          0.79                                  plant row                                                                     1990 segregation                                                                         6        10            1:2   0.72                                  for F.sub.4:5 purple                                                          plants                                                                        1990 individual                                                                          350             128    3:1   0.37                                  plant data for                                                                10 segregating                                                                rows                                                                          ______________________________________                                         .sup. Ratio = purple to pink                                                  .sup. Ratio = purple to segregating to pink?                                  .sup.§ Data in parenthesis for LN895322 and LN895320 respectively   

EXAMPLE 4 Inheritance of Pink Flower in Soybean

To test if the pink flower gene is allelic to the known flower colorgenes, a pink-flowered plant described in Stephens, P. A. and C. D.Nickell (1991) "A Pink Flower Color Mutant in Soybean," Soybean Genet.Newsl 18:226-228, was crossed to each of the five Clark flower colorisolines (Bernard, R. L. (1978) "Notice of Release of Clark and HarosoyIsolines," Soybean Genet Newsl. 1:66-75). Table 4 lists parents used inthis study and their flower colors.

                  TABLE 4                                                         ______________________________________                                        Flower color and Genotype for soybean lines used as                           parents in crosses.                                                           Parental line                                                                             Flower Color Genotype                                             ______________________________________                                        LN89-5322-2 Pink         t W1 w3 W4 Wm wp                                     Clark 63    Purple       T W1 w3 W4 Wm Wp                                     L70-4422    Purple-throat§                                                                        T W1 W3 w4 Wm Wp                                     L68-1774    Near-white   T W1 w3 w4 Wm Wp                                     L72-2181    Magenta      T W1 w3 W4 wm Wp                                     L69-4776    White        T w1 w3 W4 Wm Wp                                     Burlison    White        T w1 w3 W4 Wm Wp                                     ______________________________________                                          Each locus is considered homozygous for genes as indicated.                   T,t = tawny and gray pubescence respectively. W1, W3, W4 and Wm have bee     previously documented, Wp is our proposed designation.                        §Purplethroat = dilutepurple                                        

A pink-flowered, gray pubescence plant served as the female while Clarkisolines were used as the male parent since they possessed thephenotypic marker, tawny pubescence. A cross was confirmed by an F₁plant having tawny pubescence. To test for cytoplasmic effects,reciprocal crosses were made between the pink-flowered plant and thecultivar "Burlison" (Nickell, C. D., et al. (1990) "Registration ofBurlison Soybean," Crop Sci. 30:232) which has white flowers and tawnypubescence.

F₁ plants were grown in a greenhouse; harvested F₂ seed were grown inthe field for flower color classification. Flowers on F₂ plants wereclassified by comparing flower phenotype to flowers on Clark isolineparents. After classification for flower and pubescence color, theplants were labelled with coded plant tags. Plant tags and flower colorwere periodically reviewed to confirm proper color classification, andadditional tags were added to later flowering plants.

Table 5 presents flower color segregation data for the F₂ generation. Itcan be seen that the pink flower trait is novel, is not linked to otherflower color genes, and is not influenced by cytoplasm. In addition, a3:1 (purple:pink) ratio in the F₂ generation of LN89-5322-2 X Clark 63confirms the occurrence of a recessive allele controlling pink flowercolor. Based on the data in Table 5, the flower color genotype forLN89-5322-2 is W1W1w3w3W4W4WmWmwpwp.

                                      TABLE 5                                     __________________________________________________________________________    Soybean plants classified in the F2 generation according to their flower      color                                                                                      Total        Purple-                                                                              Near-                                               Alleles                                                                             plants                                                                            Purple                                                                            Magenta                                                                            throat                                                                            Pink                                                                             white   Theoretical                          Cross  Segr. no.                     White                                                                             ratio X.sup.2                                                                         P                            __________________________________________________________________________    LN89-5322-2x Clark 63                                                                 ##STR1##                                                                           125 88  --   --  37 --  --  3:1   1.4                                                                             0.27                         LN89-5322-2x L70-4422                                                                 ##STR2##                                                                           115 60  --   21  17  15  2  36:9:12:6:1                                                                         7.3                                                                             0.13                         LN89-5322-2x L68-1774                                                                 ##STR3##                                                                           129 75  --   --  20 27   7  9:3:3:1                                                                             1.2                                                                             0.81                         LN89-5322-2x L72-2181                                                                 ##STR4##                                                                           123 70  23   --  30 --  --  9:3:4 0 1.00                         LN89-5322-2x L69-4776                                                                 ##STR5##                                                                           111 61  --   --  15 --  45  9:3:4 3.5                                                                             0.18                         LN89-5322-2x Burlison                                                                 ##STR6##                                                                           124 73  --   --  21 --  30  9:3:4 0.4                                                                             0.88                         Burlison x LN89-5322-2                                                                ##STR7##                                                                            83 40  --   --  16 --  27  9:3:4 2.8                                                                             0.27                         __________________________________________________________________________      Since the W3w4w4wpwp phenotype is light purple throat due to the presenc     of wp, it cannot be distinguished from nearwhite and therefore                phenotypically becomes part of the nearwhite class                       

What we claim is:
 1. A soybean plant having pink flowers and uniformpigmentation throughout each petal as a distinguishing phenotype,wherein said plant is selected from the group consisting of ATCC 97108,ATCC 75579 and ATCC
 97109. 2. A plant as set forth in claim 1 whereinsaid plant additionally displays the phenotype of increased seed size ascompared to Glycine max cv. Kenwood.
 3. A plant as set forth in claim 1wherein said plant additionally displays the phenotype of increased seedprotein content as compared to Glycine max cv. Kenwood.
 4. A plant asset forth in claim 3 wherein the increased seed protein is notaccompanied by a substantial decrease in yield.
 5. A plant as set forthin claim 1 wherein said pink flower phenotype is conveyed by a recessivegene.
 6. A plant as set forth in claim 1 wherein said pink flowerphenotype co-segregates with additional phenotypes of increased seedsize and increased seed protein as compared to Glycine max cv. Kenwood.7. A plant as set forth in claim 1 having additional phenotypes of seedsize and seed protein content wherein said additional phenotypes of saidplant co-segregate with said phenotype of pink flowers, in crosses withsoybean plants lacking pink flowers, such that an F1 progeny plant ofsaid cross, when selfed yields an F2 progeny plant having pink flowersas a distinguishing phenotype and said F2 progeny plant also displaysthe additional phenotypes of seed size and seed protein content at leastequivalent to the seed size and seed protein content of said plant ofclaim
 1. 8. Seeds produced by the soybean plant of claim
 1. 9. A soybeanplant descended from a pink-flowered soybean line selected from thegroup consisting of ATCC 97108, ATCC 75579 and ATCC
 97109. 10. Thesoybean plant of claim 9 wherein said plant has pink flowers and uniformpigmentation throughout each petal as a distinguishing phenotype. 11.The soybean plant of claim 9 wherein said plant, when selfed, will yielda progeny plant having pink flowers and uniform pigmentation throughouteach petal as a distinguishing phenotype.
 12. A method of soybeanbreeding using a first parental plant having the phenotype of pinkflower color, wherein said plant is selected from the group consistingof ATCC 97108, ATCC 75579 and ATCC 97109 to introduce associated traitsinto a soybean line comprising the steps of:(a) crossing said firstparental plant with a second parental plant; (b) recovering F1 hybridplants; (c) selfing said F1 hybrid plants; (d) selecting for F2 hybridplants having said phenotype of pink flower color; and (e) back-crossingsaid pink-flowered hybrids recurrently with said first parental plant,thereby producing a soybean line having said phenotype of pink flowercolor.
 13. A method of claim 12 wherein said first parental plant is aplant of the line ATCC
 75579. 14. Seeds produced according to the methodof claim
 12. 15. A progeny plant produced according to the method ofclaim
 12. 16. A soybean plant descended from a first parental plantselected from the group consisting of ATCC 97108, ATCC 75579 and ATCC97109 and a second parental plant, wherein said plant displays thephenotype of increased seed protein content as compared to the secondparental plant.
 17. A soybean plant descended from a first parentalplant selected from the group consisting of ATCC 97108, ATCC 75579 andATCC 97109 and a second parental plant, wherein said plant displays thephenotype of increased seed size as compared to the second parentalplant.
 18. A soybean plant descended from a pink-flowered soybean lineselected from the group consisting of ATCC 97108, ATCC 75579 and ATCC97109 wherein said plant carries the flower color gene wp, and whereinsaid plant will produce progeny that have pink flowers and uniformpigmentation throughout each petal as a distinguishing phenotype.